1. Field of the Invention
The invention relates to a porous matrix, the preparation thereof, and methods of using the same.
2. Brief Discussion of the Related Art
Mass cell production is important in tissue engineering, protein drug production, and cell therapy. The conventional mass cell production technology includes static flat culturing and dynamic bioreactor culturing. Static flat culturing is appropriate for small-scale trials such as 106-108 cells cultured in laboratory or factory. To compensate for the limited surface of the culture plate, large-scale culture requires numerous culture plates, however, manual operation such as seeding cells, changing media, passaging or harvesting cells increases labor burden and the risk of contamination. It is, therefore, not economical for mass production of animal cells.
Bioreactors provide sufficient metabolic exchanges and are popular in mass production of animal cells. Bioreactors for adherent cells can be two- or three-dimensional. The former provides solid micro-beads for cells to attach to in a two-dimensional manner and also evenly distributed nutrients. Cells proliferated in this system may, however, be dedifferentiated by shear stress and friction produced by the stirring in the bioreactor, harmful to the cells. The later has porous matrices for cells to attach to in a three-dimensional manner and provides a larger space for cell growth and less shear stress, however, harvesting cells can be impeded. The proliferated cells may form an aggregate within the matrix and cannot be simply harvested by regular digesting enzyme. The longer digestion time required, however, may greatly reduce the recovery rate.
Matrices for adherent cells include natural materials such as collagen, gelatin, chitosan, or artificial material such as polyglycolide acid (PGA), polylactide acid (PLA), poly(glycolide co-lactide) acid (PLGA). These materials provide a biocompatible, biodegradable, 3-dimensional scaffold for cell attachment and proliferation and are designed to be implanted into living subjects with the culturing cells for replacement or repair of injured tissues or organ. Recovery rate is not a major issue for this application.
Materials for the production of protein drugs are not designed to be implanted into living subjects and can be artificial polymers such as polystyrene (PS), polyvinyl chloride (PVC), or polymethyl acrylate resin. For example, U.S. Pat. No. 5,254,471 discloses a carrier for cell culture comprising polyester fibers. The carrier makes it possible for the cells to retain their differentiation and proliferation ability for a long time, however, cell recovery from the carrier can be a problem. U.S. Pat. No. 5,266,476 also discloses a matrix and cultivation system for anchorage-dependent cells. The matrix provides a substantially increased available effective surface area for cell attachment by the use of non-woven or open-pore foams with suitable pore size. This matrix is suitable for cell proliferation and growth, but not appropriate for the recovery of cells.
A new matrix material is alginates, a family of unbranched polysaccharides with properties that vary widely depending on their composition. In the presence of divalent cations such as calcium ion, alginates form a gel, however, gel formation can be reversed by adding ion chelating agents such as EDTA. Recently, alginates have been used to encapsulate a variety of biological materials, including cells. For example, U.S. Pat. No. 5,585,183 discloses a method for preparing liquid-core microcapsules for cell cultures, using a hardening solution containing CaCl2 and polyethylenimine to harden alginate gel-core beads before coating them with polylysine solution and dissolving calcium ion with sodium citrate. The liquid-core microcapsules solve the problem of mechanical disruption, but the cultured cells cannot be released easily. Further, the growing space of the liquid-core microcapsules is limited.
U.S. Pat. No. 6,730,314 discloses a method for the production of chodrocytes. The method comprises encapsulating the chondrocytes in alginate beads, cultivating the encapsulated chondrocytes under a low oxygen pressure, and isolating the encapsulated chondrocytes from the alginate beads by a chelating agent for cartilage implantation. U.S. Pat. No. 5,073,491 discloses a method for cells in a bioreactor having a growth chamber for receiving the cells. The method comprises combining cells with gelatin particles to form a cell-gelatin particle suspension, mixing alginate with the cell-gelatin particles to form an alginate-cell-gelatin suspension, polymerizing the alginate to form alginate beads having the cells and gelatin particles entrapped therein, and heating the alginate beads to dissolve the gelatin particles and thereby form cavities within the alginate beads. The cavities contain the cells to be grown. In these methods, alginates are limited to encapsulating cells and the cell recovery is low or even not mentioned.
Also related to alginates are U.S. Pat. No. 5,840,777 and U.S. Pat. No. 5,723,601. The patents disclose methods of producing polysaccharide foams containing alginate by freezing and drying technology. This technology is time- and energy-consuming and cannot be used in large scale. In addition, porosity, pore size, and even cross-linking cannot be controlled. The application of cell culture is not well addressed in the two patents.
High density culture of anchorage dependent cells in carrier systems, especially the alginate system, however, is still restricted in some ways, therefore, a cell matrix providing excellent growth environment and high recovery rate is still required.